DNA helicases are DNA-stimulated ATPases that unwind duplex DNA to produce the single-stranded (ss) DNA intermediates required for replication, recombination and repair in all organisms. Our studies focus on two DNA helicases from E. coli, Rep and UvrD (Helicase H), and are designed to obtain a molecular understanding of the mechanism(s) by which DNA helicases unwind duplex DNA and translocate along DNA in reactions coupled to ATP binding and hydrolysis. Rep and UvrD function independently as helicases, but also interact to form hetero-dimers in vitro. Biochemical and biophysical approaches will be used to examine the equilibria and kinetics of the interactions that are functionally important for DNA unwinding, such as DNA and nucleotide binding and protein self-assembly (the oligomeric nature of helicases appears to be essential for DNA unwinding). Our previous DNA binding studies indicate that Rep dimerizes upon binding 55- or duplex (ds-) DNA and that nucleotides affect these interactions allosterically. An active "rolling" model for how the Rep dimer translocates and unwinds duplex DNA has been proposed that makes a number of testable predictions; many of the proposed studies are focused on testing this model. A major emphasis is on transient kinetic approaches (stopped-flow fluorescence and chemical quench-flow) to examine the kinetics and mechanism of DNA binding to and ATP binding and hydrolysis by the five Rep dimer species that differ in their DNA (ss and ds) ligation state, a subset of which appears to be intermediates in the DNA unwinding reaction. The kinetics and mechanism of nucleotide (ADP, ATP, AMPPNP and fluorescent analogs) binding, ATP hydrolysis and DNA binding will be studied using fluorescence stopped-flow and quench-flow. In parallel, we will examine Rep and UvrD-catalyzed unwinding of synthetic DNA substrates with the goal of developing a full kinetic mechanism for unwinding. Both rapid quench- flow and a novel fluorescence stopped-flow method will be used to study DNA unwinding (effects of ss-DNA tail length, ds-DNA length, sequence and base composition as well as solution conditions). The efficiency of ATP hydrolysis during unwinding and the number of base pairs unwound in a single catalytic event ("step size") will be estimated. "Passive" vs. "active" models of DNA unwinding will be tested using novel, non-natural DNA substrates. A major goal is also to determine the. active oligomeric form of the UvrD helicase and to study its interactions with DNA and nucleotides. The overall goal of these studies is to obtain basic information about the mechanism of DNA unwinding and translocation by this important class of motor proteins. However, the mechanistic information obtained should facilitate studies of other DNA and RNA helicases. Since DNA replication and repair are fundamental to cell growth in all organisms, an understanding of such a basic process as enzyme-catalyzed DNA unwinding will undoubtedly have an impact on our understanding of some cancers that result from defects in replication or repair.